Cytotoxic T lymphocytes (CTLs) play an essential role in fighting cells infected with viruses, intracellular bacteria and parasites, and tumor cells. They do so by direct cytotoxicity and by providing specific and nonspecific help to other immunocytes such as macrophages, B cells, and other T cells. Infected cells or tumor cells process antigen through intracellular events involving proteases. The processed antigen is presented on the cellular surface in the form of peptides bound to HLA class I molecules to T cell receptors on CTLs. MHC class I molecules can also bind exogenous peptides and present them to CTLs without intracellular processing.
At the present time it is difficult to accurately predict from the sequence of an antigenic protein how the protein will be processed and which peptide portions will bind HLA class I molecules and be presented to CTLs. Binding motifs have been predicted for some HLA class I molecules based on sequence analysis of peptides eluted from these molecules (Falk et al., Nature 351:290 (1991)). Further, of the peptides that are processed and do bind to HLA class I, which ones will contain CTL-recognizable epitopes is not yet predictable.
Hepatitis B Virus (xe2x80x9cHBVxe2x80x9d) is a non-lytic virus which has currently infected approximately 250 million people worldwide. HBV infection in adults typically leads to an acute disease in the majority of cases, and to a chronic disease state in a minority of patients. This ratio of acute to chronic is reversed when the infection occurs close to the time of birth. There is an increased incidence of hepatocellular carcinoma in chronic HBV infection. A small percentage of individuals who are infected with HBV in adulthood develop fulminant hepatitis associated with a strong immune response with high lethality.
While there is no effective treatment for HBV infection, vaccines have been developed in recent years to prevent HBV infection. The vaccines employ either HBV surface antigen (HBsAg) purified from the plasma of chronic HBV carriers, or HBsAg produced by recombinant DNA technology. Synthetic HBsAg peptide-based vaccines have also been proposed. See, for example, U.S. Pat. Nos. 4,599,230 and 4,599,231. The anti-HBsAg vaccines, however, afford protection in only about 90% of immunized individuals. Those who are unimmunized, or immunized but unprotected, provide a significant reservoir of potential infection.
The contribution of CTLs to immunity to HBV antigens has been difficult to assess. Chisari et al. (Microbial Pathogen. 6:31 (1989)) have suggested that liver cell injury may be mediated by an HLA-Class I restricted, CD8+ cytotoxic T cell response to HBV encoded antigens. Class I major histocompatibility (MHC)-restricted cytotoxic T lymphocyte responses have been identified for a variety of other viruses, such as influenza. For example, Townsend et al., Cell 44:959 (1986) reported that epitopes of an influenza virus nucleoprotein recognized by cytotoxic T lymphocytes could be defined by synthetic peptides. In attempting to define the cytotoxic T lymphocyte response to HBV, it has been shown that peripheral blood lymphocytes from patients with acute and chronic HBV may be able to kill autologous hepatocytes in vitro, but the specificity of the cytolytic activity, its HLA restriction elements, and cellular phenotype were not established. See, Mondelli et al., J. Immunol. 129:2773 (1982) and Mondelli et al., Clin. Exp. Immunol. 6:311 (1987). Moriyama et al., Science 248:361-364 (1990), have reported that the HBV major envelope antigen is expressed at the hepatocyte surface in a form recognizable by envelope-specific antibodies and by MHC class I-restricted, CD8+ cytotoxic T lymphocytes.
As there is a large reservoir of individuals chronically infected with HBV, it would be desirable to stimulate the immune response of these individuals to respond to appropriate HBV antigens and thereby eliminate their infection. It would also be desirable to prevent the evolution to a chronic HBV infection in individuals suffering from an acute phase infection. Further, as the presently approved HBV vaccines do not elicit protective immunity in about 10% of immunized individuals, it would be desirable to elicit more effective immunity, such as by increasing or diversifying the immunogenicity of the vaccines. Quite surprisingly, the present invention fulfills these and other related needs.
The present invention provides peptides which induce MHC class I restricted cytotoxic T lymphocyte responses against HBV antigen. The peptides of interest are derived from the sequence of the HBV polymerase protein. In certain embodiments the CTL inducing peptide will have the sequence of HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No. 2]; HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No. 20]; HBpol748-757 (Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg) [Seq ID No. 21]; HBpol758-766 (Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) [Seq ID No. 22]; HBpol773-782 (Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val) [Seq ID No. 3]; HBpol803-811 (Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; and HBpol816-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5]; or will have a sequence substantially homologous to one of the foregoing sequences. The peptide can be optionally flanked and/or modified at one or both of the N-and C-termini, as desired. Conservative substitutions, deletions and additions may be made at non-critical residue positions within the selected peptide without substantially adversely affecting its biological activity.
The present invention provides peptides which induce MHC Class I restricted cytotoxic T lymphocyte responses against HBV antigen. The peptides of interest are derived from the HBV nucleocapsid. In certain embodiments the peptides comprises from six to about fifty amino acids and have at least four contiguous amino acids from within the sequence (HBc19-27) Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val, [SEQ ID NO:68] wherein the peptide can be optionally flanked and/or modified at one or both of the N- and C-termini, as desired. In preferred embodiments the peptide sequences are Ala-Thr-Val-Glu-Leu-Leu-Ser-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val, (HBc11-27) [SEQ ID NO:67], and Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val (HBc19-27) [SEQ ID NO:68], which regions are conserved among the major subtypes of HBV, although substitutions, deletions and additions may be made to the peptide without substantially adversely affecting its biological activity.
In other embodiments the peptides are from six to about fifty amino acids and have at least four contiguous residues from with the sequence (HBc140-154) Leu-Ser-Thr-Leu-Pro-Glu-Thr-Thr-Val-Val-Arg-Arg-Arg-Gly-Arg [SEQ ID NO:71], wherein the peptide can be optionally flanked and/or modified at one or both of the N- and C-termini, as desired. In other embodiments the peptides are derived from at least seven contiguous amino acids of the sequence of HBc111-125, wherein HBc111-125 for HBV subtype awy has the sequence Gly-Arg-Glu-Thr-Val-Ile-Glu-Tyr-Leu-Val-Ser-Phe-Gly-Val-Trp [SEQ ID NO:70], wherein termini may be also modified as mentioned above. For the HBV subtype adw, the Ile116 is replaced by Leu. In yet another embodiment a peptide which induces a MHC Class I-restricted cytotoxic T lymphocyte response is peptide (HBc28-47) [SEQ ID NO:72] having the sequence Arg-Asp-Leu-Leu-Asp-Thr-Ala-Ser-Ala-Leu-Tyr-Arg-Glu-Ala-Leu-Glu-Ser-Pro-Glu-His, wherein termini of the selected peptide can also be modified, as desired.
In the various peptide embodiments it will be understood that the peptides can be polymerized, each to itself to form larger homopolymers, or with different peptides to form heteropolymers. In some instances peptides will be combined in a composition as an admixture and will not be linked. The peptide can also be conjugated to a lipid-containing molecules capable of enhancing a T lymphocyte response, or to a different peptide which induces a T-helper cell response, for example.
Compositions are provided which comprise a peptide of the invention formulated with an additional peptide, a liposome, an adjuvant and/or a pharmaceutically acceptable carrier. Thus, pharmaceutical compositions can be used in methods of treating acute HBV infection, particularly in an effort to prevent the infection from progressing to a chronic or carrier state. Methods for treating chronic HBV infection and HBV carrier states are also provided, where the pharmaceutical compositions are administered to infected individuals in amounts sufficient to stimulate immunogenically effective cytotoxic T cell responses against HBpol epitopes. For treating these infections it may be particularly desirable to combine the peptides which induce MHC class I restricted cytotoxic T lymphocyte responses against HBV antigen with other peptides or proteins that induce immune response to other HBV antigens, such as HBV envelope or core. To treat individuals with chronic or carrier state infections the compositions may be administered in repeated dosages over a prolonged period of time, as necessary, to resolve or substantially mitigate the infection and/or shedding of virus.
Vaccine compositions for preventing HBV infection, particularly chronic HBV infection, are also provided. The vaccine compositions comprise an immunogenically effective amount of a HBV polymerase peptide mentioned above which induces a MHC class I restricted cytotoxic T lymphocyte response, such as HLA-A2, -A1, -A3, A-11, and/or -A24, and will typically further comprise an adjuvant, e.g., incomplete Freund""s adjuvant or aluminum hydroxide. To achieve enhanced protection against HBV, the vaccine can further comprise components which elicit a protective antibody response to other HBV antigen, such as envelope (surface) antigen.
In yet other embodiments the invention relates to methods for diagnosis, where the peptides of the invention are used to determine the presence of lymphocytes in an individual which are capable of a cytotoxic T cell response to HBV polymerase antigen. The absence of such cells determines whether the individual of interest is susceptible to developing chronic HBV infection. Typically the lymphocytes are peripheral blood lymphocytes and the individual of interest is suffering from an acute HBV infection.